PROJECT SUMMARY The overall goal of this grant proposal is to better understand the role of gap junctions, connexin hemichannels and calcium-activated chloride channels in maintaining lens homeostasis and to determine how perturbations in these systems lead to the development of cataracts and the inability to focus accurately. These channels appear to regulate the clarity, volume, and shape of the individual lens fiber cells that make up the intact lens. A lack of proper flow patterns in the lens leads to cataracts. Problems in these lens systems also likely affect the ability of the lens to alter its shape during accommodation. This problem is particularly acute in individuals suffering from presbyopia. There are three specific aims: Aim 1: To characterize the effects of cataract-associated connexin mutations using a combination of experimental and modeling approaches. We will focus on cataract associated mutations located in regions that are known to be important in the gating of undocked hemichannels. These studies will help to identify mechanisms by which the mutations lead to disease. They may also identify potential targets for the development of therapeutic agents to modulate the open probability of connexin channels. Aim 2: To use a new preparation developed by our laboratory to gain a better understanding about how fiber cells respond to mechanical stress. The following hypothesis will be tested: (1) Cx46 hemichannels are activated by mechanical stress. The proposed studies will examine the mechanosensitivity of these channels in peripheral fiber cells lacking Cx50 using the whole cell patch clamp technique. Mechanical stretch will be induced using two different stimuli: (1) positive pressure pulses applied to the patch pipette; (2) hypotonic stress. The possible contribution of other channels to the mechanosensitive membrane conductance in lens fiber cells will be investigated by performing whole cell patch clamp experiments on double knockout (Cx46-/- ;Cx50-/-) fibers. Aim 3: To understand the role of calcium-activated chloride channels in volume regulation of peripheral fiber cells. The following two hypotheses will be tested: (3a) peripheral lens fiber cells express calcium-activated chloride channels that can be attributed to the anion channel protein, TMEM16a; (3b) these channels can be indirectly activated by membrane stretch via purinergic signaling or calcium influx through mechanosensitive channels.